Overexpression of HER2, a receptor tyrosine kinase (RTK) is found in a series of cancers, most notably, 30% of breast cancers and 10% of ovarian cancers. Elevated levels of the receptor are associated with constitutive receptor activation, cellular transformation, and poorer survival of patients in a clinical setting. Activation of RTKs is known to involve dimerization. HER2, which has a potent cytoplasmic kinase domain but fails to bind any ligand by itself, heterodimerizes with other RTKs, preferentially with the kinase-deficient but ligand-binding HER3. The preferred ligand for the heterodimer is heregulin. However, at elevated levels, full-length and membrane-bound HER2 can self-associate ligand independently. The relative contribution of ligand-independent self-association versus an overall increase in ligand-dependent heterodimerization is an unresolved question for HER2 overexpressing cancers. A key objective of our work is to dissect those two reactions on a molecular level. To this end, we will identify and mutate the self association interface of HER2. We found recently that the catalytically inactive HER3, as well as its soluble extracellular domain (ECD), self-associates at low concentrations, a reaction that is disrupted by ligand binding. We will use the strong self-association of HER3 and its high homology with HER2 to identify self association sites in both receptors. This identification relies on computational predictions by 3D cluster analysis as well as alternative mutagenesis and selection approaches. We will apply FRET and DHFR fragment complementation to obtain direct measurements of the extent of receptor association in intact cells. We will use these methods to confirm the identified regions in HER3 and HER2 and evaluate the correlation between the concentration-dependent receptor self-association of HER2 and constitutive signaling and the enhancement of tumor formation. In addition we have evidence that HER3 self-association may modulate heregulin signaling. We will evaluate the possible regulatory role of HER3 self-association and its impact on ligand-dependent and independent signaling by HER2. A better understanding of the specific receptor interactions at elevated expression levels will enhance our understanding of cellular transformation by HER2, help understand and improve the action of drugs like Herceptin, and identify specific and measurable association events as targets for drug development.